Bottom fluorescence illumination assembly for an imaging apparatus

CROSS-REFERENCE TO RELATED APPLICATION

0001This application is a continuation-in-part of a U.S. patent application entitled “FLUORESCENCE ILLUMINATION ASSEMBLY FOR AN IMAGING APPARATUS” by Nilson et al., filed Feb. 20, 2002, U.S. application Ser. No. 10/081,040, which in turn claims priority under 35 U.S.C. 119(e) from U.S. Provisional Patent Application No. 60/359,663, entitled same and filed Feb. 22, 2002, both of which are incorporated herein by reference for all purposes.

TECHNICAL FIELD

0002The present invention relates generally to imaging systems, and more particularly, relates to macroscopic, bottom fluorescent illumination sources and their associated components to illuminate imaging systems.

BACKGROUND

0003One specialized type of imaging involves the capture of low intensity fluorescence. Briefly, fluorescence is a molecular phenomenon in which a substance absorbs light of a particular wavelength and emits light of a longer wavelength. The absorption of light is referred to as the “excitation”, and the emission of longer wave lights as the “emission”. Both organic and inorganic substances can exhibit fluorescent properties.

0004Fluorescence imaging is performed by illuminating a sample to excite fluorescence molecules in the sample, and then capturing an image of the sample as it fluoresces using a camera. Such imaging applications present particular challenges to the design of a box or chamber in which the sample is contained during imaging. This is especially true in macroscopic applications where the field-of-view is about 10 cm-30 cm in diameter, as compared to microscopic applications where the field-of-view is less than about 1 cm.

0005Typically, intensified or cooled charge-coupled device (CCD) cameras are used to detect the fluorescence of low intensity light radiating from the sample. These cameras are generally complex, may require specialized cooling, and are typically fixed to a single location on the top of a specimen chamber. A user places a sample at a predetermined position in the specimen chamber within the field of view for the overhead camera.

0006Due to this static design, one particular challenge to imaging apparatus design is the diverse lighting needs required during image capture. Fluorescent image capture, of course, involves the sample being illuminated with an in-box illumination source, while the minute amounts of fluoresced from the “excited” sample are detected using a light detector, e.g., a CCD camera.

0007One problem associated with the capture of overhead images in macroscopic applications is that the relatively large CCD camera is typically centrally located directly over the sample platform which supports the sample. A single illumination source is thus often positioned in the light box at a location off-set from the camera lens, and angularly directed at the sample platform. Thus, for relatively non-planar samples supported atop the platform, substantially uniform illumination is difficult to achieve. Such is also the case when multiple illumination sources are applied which often causes detrimental shadowing, and thus, non-uniform lighting.

0008Another problem associated with fluorescent imaging in macroscopic applications is that the current imaging apparatus generally employ dichroic mirrors to perform partial filtering functions. Briefly, dichroic mirrors are typically used in fluorescence microscopes to provide an additional amount of separation for the excitation and emission wavelengths. The dichroic mirror is usually mounted at about a 45 degree angle to excitation and emission light. The excitation light is reflected by the dichroic mirror onto the specimen, while the emission light passes through the dichroic mirror, the emission filter, the lens, and is incident on the CCD camera. Dichroic mirrors are commonly used on microscopes because the beam size is very small and so the mirrors are quite compact (usually 1 inch or less in diameter).

0009For a macroscopic application, as mentioned, the required field-of-view is much larger (i.e., 10 cm-30 cm) than that for a microscopic application (less than about 1 cm). This of course necessitates the use of a much larger lens which in turn renders the use of a dichroic mirror impractical. Due to the size and orientation of such a mirror in the imaging compartment of the imaging box, the footprint of the imaging box is unfeasibly large. In view of the foregoing, an improved illumination assembly for a light box that enables the substantially uniform lighting for fluorescent image capture of the sample would be highly desirable.

DISCLOSURE OF INVENTION

0010The present invention provides a macroscopic fluorescence illumination assembly for use with an imaging apparatus with a light-tight imaging compartment. The imaging apparatus includes an interior wall defining a view port extending into the imaging compartment to enable viewing of a specimen contained therein. The illumination assembly includes a specimen support surface sized and dimensioned for receipt in the imaging compartment, and oriented to face toward the view port of the imaging apparatus. The support surface is substantially opaque and defines a window portion that enables the passage of light there through. The window portion is selectively sized and dimensioned such that the specimen, when supported atop the support surface, can be positioned and seated over the window portion in a manner forming a light-tight seal substantially there between. The illumination assembly further includes an excitation light source, and a bundle of fiber optic strands having proximal ends thereof in optical communication with the light source. The distal ends of the strands terminate proximate the window portion of the support surface. The distal ends each emit a respective beam of light originating from the light source which are then collectively directed toward the window portion and into a bottom side of the specimen wherein the diffused light passes there through and exits a topside thereof for receipt through the view port to view the fluorescence of the specimen.

0011Accordingly, by illuminating the specimen through a bottom side illumination thereof with an excitation light source, as opposed to a topside illumination of the specimen, the autofluorescence background signal of the specimen itself is reduced. This is due to the fact that tissue autofluorescense is always higher on the side of the excitation light source than on the side facing the camera. In the case of a topside illumination, both the camera and the excitation light source are on the same side.

0012In one specific arrangement, a specimen illumination platform is provided including a cover plate that provides the support surface, and a support structure cooperating with the cover plate to define a light-tight interior cavity below the window portion. The distal ends of the fiber optic strands terminate in this cavity. The substantially parallel optical axes of the distal ends of the fiber optic are oriented in the cavity substantially perpendicular to a plane containing the window portion of the support surface. A reflector device is disposed in the cavity, and includes a substantially planar reflective surface oriented at an angle about 45° relative the direction substantially parallel to the optical axes of the distal ends of the fiber optic strands. In this orientation, the directional beams of light emitted from the fiber optic distal ends are reflected through the window portion and into the specimen. The distal ends of the fiber optic bundle are aligned in a linear array in another embodiment extending substantially along the elongated reflective surface.

0013The size window portion of the cover plate may be selected according to the specimen. It is preferably substantially rectangular shaped, and may be provided by a void in the cover plate or a transparent material.

0014In another configuration, a macroscopic fluorescence imaging assembly is provided for viewing a specimen. The imaging assembly includes an imaging apparatus having an enclosure wall defining a view port into a light-tight imaging compartment containing the specimen thereof, and a specimen illumination platform positioned in the imaging compartment having a support surface facing toward the view port. The support surface is substantially opaque and defines a window portion enabling the passage of light there through. The window portion is selectively sized and dimensioned such that the specimen, when supported atop the support surface, can be positioned and seated over the window portion in a manner forming a light-tight seal substantially there between. An illumination device is disposed in the imaging compartment below the specimen illumination platform and proximate the window portion of the support surface such that light emitted from the illumination device is directed toward the window portion and into a bottom side of the specimen wherein the diffused light exits a topside thereof for receipt through the view port to view the fluorescence of the specimen.

BRIEF DESCRIPTION OF THE DRAWING

0015The assembly of the present invention has other objects and features of advantage which will be more readily apparent from the following description of the best mode of carrying out the invention and the appended claims, when taken in conjunction with the accompanying drawing, in which:

0016<figref idref="DRAWINGS">FIG. 1</figref> is a top perspective view of an imaging apparatus, with the door removed, incorporating an illumination assembly constructed in accordance with the present invention.

0017<figref idref="DRAWINGS">FIG. 2</figref> is a bottom perspective view of the imaging apparatus of <figref idref="DRAWINGS">FIG. 1</figref>, and illustrating a light ring component of the illumination assembly.

0018<figref idref="DRAWINGS">FIG. 3</figref> is a front elevation view of the illumination assembly of <figref idref="DRAWINGS">FIG. 1</figref> incorporated in the imaging apparatus.

0019<figref idref="DRAWINGS">FIG. 4</figref> is a side perspective view of a light source and a filter wheel assembly of the illumination assembly of <figref idref="DRAWINGS">FIG. 1</figref> optically coupled to the imaging apparatus.

0020<figref idref="DRAWINGS">FIG. 5</figref> is an enlarged, bottom plan view of the light ring component of the of the illumination assembly of FIG. <b>1</b>.

0021<figref idref="DRAWINGS">FIG. 6</figref> is an enlarged, side elevation view, in cross-section, of the filter wheel assembly of the illumination assembly of FIG. <b>1</b>.

0022<figref idref="DRAWINGS">FIG. 7</figref> is an enlarged, exploded, side elevation view, in cross-section, of the plate members of a baffle device of the filter wheel assembly of FIG. <b>6</b>.

0023<figref idref="DRAWINGS">FIG. 8</figref> is an enlarged, fragmentary side elevation view, in cross-section, of an optical connection assembly of the illumination assembly of FIG. <b>1</b>.

0024<figref idref="DRAWINGS">FIG. 9</figref> is an enlarged, side elevation view, in cross-section, of the optical connection assembly of FIG. <b>8</b>.

0025<figref idref="DRAWINGS">FIG. 10</figref> is an enlarged, bottom plan view of the light ring component of the of the illumination assembly of <figref idref="DRAWINGS">FIG. 1</figref>, and illustrating a second light filter thereof in phantom lines.

0026<figref idref="DRAWINGS">FIG. 11</figref> is a side elevation view, in cross-section, of the light-tight seal mechanism and second filter wheel taken substantially along the plane of the line <b>11</b>—<b>11</b> in FIG. <b>10</b>.

0027<figref idref="DRAWINGS">FIG. 12</figref> is an enlarged, bottom perspective view of the imaging apparatus of <figref idref="DRAWINGS">FIG. 1</figref>, and illustrating an alternative embodiment illumination assembly.

0028<figref idref="DRAWINGS">FIG. 13</figref> is an enlarged, bottom perspective view of the alternative embodiment illumination assembly of <figref idref="DRAWINGS">FIG. 12</figref> mounted to the upper interior wall of the imaging apparatus.

0029<figref idref="DRAWINGS">FIG. 14</figref> is a bottom plan view of the alternative embodiment illumination assembly of FIG. <b>12</b>.

0030<figref idref="DRAWINGS">FIG. 15</figref> is an enlarged, bottom perspective view of a light dispersion assembly of the illumination assembly of FIG. <b>12</b>.

0031<figref idref="DRAWINGS">FIG. 16</figref> is an enlarged, bottom perspective view of the of the light dispersion assembly of FIG. <b>12</b>.

0032<figref idref="DRAWINGS">FIG. 17</figref> is an enlarged, bottom perspective view of the alternative embodiment illumination assembly of <figref idref="DRAWINGS">FIG. 12</figref> illustrating the protective cover device shown in phantom lines.

0033<figref idref="DRAWINGS">FIG. 18</figref> is a rear perspective view of a light source and a filter wheel assembly of the illumination assembly of <figref idref="DRAWINGS">FIG. 12</figref> optically coupled to the imaging apparatus.

0034<figref idref="DRAWINGS">FIG. 19</figref> is an enlarged, side elevation view, in cross-section, of the filter wheel assembly of the illumination assembly mounted to the imaging apparatus of FIG. <b>18</b>.

0035<figref idref="DRAWINGS">FIG. 20</figref> is a top perspective view of an alternative embodiment bottom illumination assembly providing bottom illumination of the specimen constructed according to the present invention.

0036<figref idref="DRAWINGS">FIG. 21</figref> is an enlarged, top perspective view of the bottom illumination assembly of <figref idref="DRAWINGS">FIG. 20</figref> without the specimen thereatop.

0037<figref idref="DRAWINGS">FIG. 22</figref> is an enlarged, top perspective view of the bottom illumination assembly of <figref idref="DRAWINGS">FIG. 20</figref> with the cover plate removed.

0038<figref idref="DRAWINGS">FIG. 23</figref> is an enlarged, top perspective view of the bottom illumination assembly of <figref idref="DRAWINGS">FIG. 20</figref> with a slide device positioned thereatop.

0039<figref idref="DRAWINGS">FIG. 24</figref> is an exploded top perspective view of the bottom illumination assembly of FIG. <b>20</b>.

BEST MODE OF CARRYING OUT THE INVENTION

0040While the present invention will be described with reference to a few specific embodiments, the description is illustrative of the invention and is not to be construed as limiting the invention. Various modifications to the present invention can be made to the preferred embodiments by those skilled in the art without departing from the true spirit and scope of the invention as defined by the appended claims. It will be noted here that for a better understanding, like components are designated by like reference numerals throughout the various figures.

0041Referring now to <figref idref="DRAWINGS">FIGS. 1-5</figref>, a fluorescence imaging assembly, generally designated <b>20</b>, is provided which includes a light-tight sample box or imaging apparatus <b>21</b> having an enclosure wall or upper housing <b>22</b> defining a view port <b>23</b> (<figref idref="DRAWINGS">FIG. 5</figref>) into a light-tight imaging compartment <b>25</b> thereof. A specimen platform <b>26</b> is positioned in the imaging compartment <b>25</b> which includes a support surface <b>27</b> facing toward the view port <b>23</b>. The imaging assembly <b>20</b> further includes an illumination assembly, generally designated <b>28</b>, having an illumination device <b>30</b> disposed in the imaging compartment <b>25</b>, and positioned proximate to and substantially peripherally encircling the view port <b>23</b> such that said specimen platform <b>26</b> is illuminated in a substantially uniform manner.

0042Accordingly, by illuminating the specimen platform <b>26</b> from an illumination device peripherally extending around or continuously surrounding the camera view port <b>23</b>, a specimen (not shown) positioned on the platform <b>26</b> will be substantially uniformly illuminated, symmetrically about the optical axis. Such uniform illumination is not attainable in the off-set lighting techniques currently applied without repositioning the lighting or the specimen.

0043Moreover, as will be apparent below, the improved filtering performance, the careful selection of low auto fluorescent materials for the filters and lighting components, as well as the formation of light tight seals essentially from the light source to the imaging compartment of the imaging apparatus, collectively enable sufficient filtered fluorescent lighting without the need for additional filtering through dichroic mirrors. Thus, in this macroscopic fluorescent imaging application, these relatively large dichroic filters can be eliminated. Consequently, the overall footprint of the imaging enclosure is substantially reduced.

0044Briefly, <figref idref="DRAWINGS">FIGS. 1-4</figref> illustrate an imaging apparatus <b>21</b> suitable for capturing photographic, fluorescent or luminescence images in accordance with one embodiment of the present invention. The imaging apparatus <b>21</b> includes an upper housing <b>22</b> defining the view port in which a lens system of a high sensitivity camera <b>31</b> is mounted. This camera is preferably an intensified or cooled integrating Charge-Coupled Device (CCD) camera <b>31</b> which is positioned on top of the imaging apparatus <b>21</b> and positioned above the upper housing <b>22</b>. The CCD camera <b>31</b> is capable of capturing fluorescent, luminescent and photographic (i.e., reflection based images) images of the sample within the imaging apparatus <b>21</b>. The CCD camera <b>31</b> may be cooled by a suitable source such as a refrigeration device that cycles a cryogenic fluid through the CCD camera via conduits. A suitable refrigeration device is the “CRYOTIGER” compressor, which can be obtained from IGC-APD Cryogenics Inc., Allentown, Pa. Other methods, such as liquid nitrogen, may be used to cool the CCD camera <b>31</b>.

0045An image processing unit optionally interfaces between camera <b>31</b> and a computer through cables. The computer, which may be of any suitable type, typically comprises a main unit that contains hardware including a processor, memory components such as random-access memory (RAM) and read-only memory (ROM), and disk drive components (e.g., hard drive, CD, floppy drive, etc.). The computer also includes a display and input devices such as a keyboard and mouse. The computer is in communication with various components in the imaging apparatus <b>21</b> via cable. To provide communication and control for these components, the computer includes suitable processing hardware and software configured to provide output for controlling any of the devices in the imaging apparatus <b>21</b>. The processing hardware and software may include an I/O card, control logic for controlling any of the components of the imaging assembly <b>20</b>, and a suitable graphical user interface for the imaging assembly <b>20</b>. The computer may also include suitable processing hardware and software for the camera <b>31</b> such as additional imaging hardware, software, and image processing logic for processing information obtained by the camera <b>31</b>. Components controlled by the computer may include the camera <b>31</b>, the motors responsible for camera <b>31</b> focus, the motors responsible for position control of a platform supporting the sample, the camera lens, filter wheels, f-stop, etc. The logic in computer may take the form of software, hardware or a combination thereof. The computer also communicates with a display for presenting imaging information to the user. By way of example, the display may be a monitor, which presents an image measurement graphical user interface (GUI) that allows the user to view imaging results and also acts as an interface to control the imaging assembly <b>20</b>.

0046Referring now to <figref idref="DRAWINGS">FIGS. 2</figref>, <b>4</b> and <b>5</b>, the illumination assembly <b>28</b> includes a frame <b>32</b> supporting the illumination device <b>30</b> which is mounted to the upper housing through a nut plate <b>33</b>. The frame <b>32</b> is preferably a rigid, ring-shaped structure having an interior diameter slightly larger than that of the view port <b>23</b> (<figref idref="DRAWINGS">FIG. 5</figref>) so as to peripherally surround it without obstructing the view from the lens system. Although the illustrated illumination device and the supporting frame <b>32</b> are circular, other geometric forms may be applied as long as the illumination device extends generally around the view port <b>23</b>.

0047In one specific embodiment, the illumination device is provided by a fiber optic lighting system having a plurality or bundle <b>35</b> of fiber optic strands extending into the imaging compartment <b>25</b>. The proximal ends <b>36</b> of the strands of the bundle <b>35</b> are positioned in optical communication with a light source <b>37</b> to transmit collected light to the distal ends <b>38</b> of the fiber optic strands. To optimize the system for use fluorescent image capture in accordance with the present invention, the material composition of the fiber optic strands are selected to have low auto-fluorescence properties. All materials (glasses, etc.), it will be understood, will fluoresce at some level. Ordinary optical glasses (E.g., float glass, BK7) contain impurities that can fluoresce. Although the autofluorescence of glass is fairly low, the extremely sensitive cameras utilized in the present invention will easily detect the autofluorescence of these materials. The glass (or other material) autofluorescence passes through the emission filter and creates noise in the fluorescence signal, so every effort is made to minimize autofluorescence. One material particularly suitable for the fiber optic strands and filters is high purity fused silica, such as plastic clad fused silica or silica clad fused silica, which has very low autofluorescence.

0048As shown in <figref idref="DRAWINGS">FIG. 5</figref>, a bottom face <b>40</b> of the frame <b>32</b> defines an annular slit <b>41</b> upon which the distal ends <b>38</b> of the strands terminate at a position generally perpendicular to the face <b>40</b>. Accordingly, the plurality of distal ends <b>38</b> of the fiber optic strands each independently emit a conical directional beam of light (illustrated by broken lines <b>39</b>) onto the specimen platform.

0049Preferably, a collective cone of light (illustrated by broken lines <b>39</b>) is emitted having with a full angle of about 55°, wherein each strand emits light in the range of about 50° to about 60°. By positioning the strand distal ends <b>38</b> at least about 140 mm to about 380 mm from the specimen platform, the collective ring of conical light beams emitted from strand distal ends <b>38</b> sufficiently overlap (<figref idref="DRAWINGS">FIG. 3</figref>) to produce relatively uniform illumination of the specimen. That is, the illumination intensity does not vary by more than about ±25% over the entire field of view, which is between about 10 cm to about 25 cm in these macroscopic applications. Moreover, these diverging beams, as will be discussed below, are substantially directed onto the specimen platform to illuminate only the specimen, and to reduce detrimental florescence of the other box components in the imaging compartment.

0050The strand distal ends are positioned substantially continuously around the annular slit <b>41</b>, and are about 3-6 strands wide. It will be appreciated, however, that complete continuity of the strand distal ends is not required to provide uniform lighting in accordance with the present invention. One example of such ring light guides is model 70001148001 by Dolan Jenner Industries of Lawrence, Mass. These fixtures are typically utilized in non-light-tight microscopic applications. Accordingly, these applications are non-fluorescent in nature.

0051To protect the exterior fiber optic bundle portions <b>50</b> and <b>51</b> of the fiber optic bundle <b>35</b> and to reduce the introduction of exterior light or noise into the fiber optic strands, a segmented, flexible metal jacket (not shown) is placed around these portions which is further surrounded by a flexible PVC sleeve <b>42</b>. This sleeve is opaque (black), and blocks all external ambient light that might enter the fiber bundle.

0052While this protective sleeve combination is sufficient to substantially reduce the introduction of exterior noise into the fibers (i.e., for use in the exterior bundle portions <b>50</b> and <b>51</b> outside the of imaging box), it may be phosphorescent and is therefore unsuitable for use inside the box since, in these low-intensity imaging applications, even an amount of light emanating from within the protective sleeve will detrimentally influence the fluorescent imaging of the specimen.

0053Accordingly, internal to the imaging box, the sleeve material <b>43</b> surrounding the bundle portion <b>53</b> of the fiber optic bundle <b>35</b> is replaced with a non-phosphorescent material to substantially eliminate the possibility of spurious light sources from the fiber optic bundle <b>35</b> within the imaging box. One particular flexible material which has low phosphorescence is a polyolefin heat shrink tubing material.

0054The proximal end <b>36</b> of the fiber optic bundle <b>35</b> is coupled to a fiber optic light source <b>37</b> which optically couples the proximal end faces of the fiber optic strands (not shown) with a direct light of the light source. Preferably, the light source includes a housing <b>45</b> which provides a connector <b>46</b> to position the proximal end faces of the fiber optic strands substantially adjacent the light source so that the light can be transmitted through the fiber optic strands of the bundle <b>35</b>. One example of such a Fiber Optic Illuminator is model PL 900, by Dolan Jenner Industries of Lawrence, Mass.

0055In one embodiment, the direct light is provided by a bulb contained in the housing <b>45</b>, and positioned at the proximal end faces of the fiber optic strands. A preferred light comprises a tungsten halogen lamp, which emits a wide spectrum of bright white light suitable to fluoresce objects. Other applicable light sources include xenon lamps, mercury lamps and lasers.

0056Typically, the usable fluorescence spectrum is in the range of 400 nm to about 900 nm. Thus, depending upon the desired fluorescence spectra, the composition of the sample material and the fluorescent material, the remaining light emitted by the light source must be filtered out. Optical filters are applied, accordingly, to filter out non-fluorescence spectra as well as unwanted fluorescence spectra. Depending upon the application, there have been selected optical filters or filter wheels disposed in the imaging compartment of an imaging apparatus <b>21</b> just after the off-set light source. Such an arrangement, however, would not be practical in the lighting technique of the present invention since the diameter of the ring-shaped frame <b>32</b> is significantly larger. Moreover, proportionate to the size of the imaging compartment, a filter wheel could not be deployed.

0057In accordance with the present invention, a filter wheel assembly, generally designated <b>47</b>, is positioned “in-line” in the fiber optic bundle <b>35</b> (<figref idref="DRAWINGS">FIGS. 1</figref>, <b>4</b> and <b>6</b>). Preferably, the filter wheel assembly <b>47</b>, which includes a plurality of optical filters, is positioned in close proximity to the transmission box. This enables the filter wheel assembly and the transmission box to be supported on a common support frame <b>48</b>, and to be packaged together as a single unit.

0058Briefly, as best illustrated in <figref idref="DRAWINGS">FIGS. 2</figref>, <b>4</b> and <b>5</b>, the fiber optic bundle <b>35</b> includes a first bundle portion <b>50</b>, extending between the light source <b>37</b> and the filter wheel assembly <b>47</b>, and a second bundle portion <b>51</b>, extending between the filter wheel assembly <b>47</b> and an optical connector assembly <b>52</b> mounted to the imaging apparatus <b>21</b>. Finally, the fiber optic bundle includes a third bundle portion <b>53</b> extending from the optical connector assembly <b>52</b> (as will be described in greater detail below) on the inside of the imaging compartment <b>25</b> to the frame <b>32</b>. This third bundle portion <b>53</b>, as above-mentioned, includes the heat shrink material sleeve <b>43</b> which has low phosphorescence.

0059The optical filters are typically interference-type filters which include bandpass filters, longpass filters and shortpass filters. These filters are preferably provided as a filter set contained on a filter wheel <b>55</b> of the filter wheel assembly <b>47</b> which is placed in-line with the fiber optic bundle <b>35</b>. Thus, the filter wheel <b>55</b>, rotatably mounted in a recess <b>56</b> of the housing <b>57</b>, can be selectively rotated to position the selected filter in the path of the fiber optic strands.

0060Each interference filter is selected to allow the passage of select spectra of light. Another useful parameter, in accordance with the present invention, is selecting a filter with sharp cut-offs or edges so that the gap between the excitation and emission filter bands can be minimized. This is beneficial in that the Stokes shift for many common fluorescent dyes and proteins is relatively small. The Stokes shift is the separation in wavelength between the excitation and emission peak. Usually, a filter gap of about 20 nm is chosen. These filters also are characterized by very high rejection outside the passband or “out-of-band” blocking properties, with a typical rejection of >10⁶. Furthermore, the filters are also preferably constructed from low auto-fluorescent materials. Accordingly, the application of these higher quality excitation and emission filters allows the use of a single excitation-emission filter pair without an associated dichroic filter.

0061Examples of such filters include the Alpha Technology filters from Omega Optical, Inc. of Brattleboro, Vt. For a general discussion on fluorescence and filtering, see the <i>Handbook of Optical Filters for Fluorescence Microscopy</i>; by Jay Richman of the Chroma Technology Corp, June 2000, and herein incorporated by reference in its entirety. The housing <b>57</b> of the filter wheel assembly <b>47</b> is substantially light-tight so that detrimental exterior light is not introduced as the light is transmitted through the filters. The housing, as viewed in <figref idref="DRAWINGS">FIG. 6</figref>, further includes an input port <b>58</b> and an output port <b>60</b> upon which the selected filter optically aligns therewith for the filtering of the light. Accordingly, a first connector <b>61</b> is included which is adapted to optically align an optical output end <b>62</b> of the first bundle portion <b>50</b> within the input port <b>58</b> of the housing for transmission of the light through the filter <b>63</b>. Similarly, the filter wheel assembly <b>47</b> includes a second connector <b>65</b> which is adapted to optically align an optical input end <b>66</b> of the second bundle portion <b>51</b> within the output port <b>60</b> of the housing for reception of the filtered light from the filter <b>63</b>.

0062To facilitate transmission of the light through the filter, a collimating lens <b>67</b> is positioned in the input port <b>58</b> between the optical output end <b>62</b> of the first bundle portion <b>50</b> and the filter <b>63</b>. In order for the excitation filter to function properly, the light rays must be fairly well collimated (parallel to the optical axis) through the filter. Therefore, as the light passes through the collimating lens, it is collimated in a direction substantially perpendicular to the planar face of the filter which minimizes detrimental reflection there from. Further, by selecting the first bundle portion <b>50</b> of the fiber optic bundle <b>35</b>, extending between the light source <b>37</b> and the filter wheel assembly <b>47</b>, to be about ¼ inch in diameter, most of the exiting light rays have a maximum cone angle in the range of about 30° to about 40°. Consequently, after passing through the collimating lens <b>67</b>, the angle of incidence is reduced to a maximum ray angle of less than or equal to about 12° The output of the excitation filter/lens assembly couples into the ½ inch diameter fiber optic bundle portion <b>51</b> in order to mate up with the ring light, which also as a ½ inch bundle size.

0063A focusing lens <b>68</b> is further disposed downstream from the filter <b>63</b> to focus and direct the collimated and filtered light, exiting the filter <b>63</b>, into the optical input end <b>66</b> of second bundle portion <b>51</b> for transmission through the fiber optic strands thereof. <figref idref="DRAWINGS">FIG. 6</figref> best illustrates that the focusing lens <b>68</b> is positioned in the output port <b>60</b> between the filter <b>63</b> and the optical input end <b>66</b> of the second bundle portion <b>51</b> of the fiber optic bundle <b>35</b>. Typical of these filter wheel assemblies, by way of example, is model FA-448, by Acton Research of Acton, Mass. It will be appreciated, however, that light-tight filter cassettes and filter bars may be employed as well.

0064While the collective optical arrangement of a conventional filter wheel assembly is applicable for most optical applications, this set-up is not suitable for fluorescent imaging. This is due to the fact that trace amounts of unfiltered light often leak around the periphery of the filter which detrimentally affect the fluorescent imaging of the sample in the imaging compartment. Although the housing <b>57</b> of these conventional filter wheel assemblies is considered light-tight, when the angle of transmission of some rays of light exiting the optical output end <b>62</b> of the first bundle portion <b>50</b> are sufficiently skewed from a direction parallel to the optical axis of the output end, unfiltered light can pass along the outer edges of the collimating lens <b>67</b>, and thus, past the filter <b>63</b> and into the focusing lens.

0065The skewing of the light rays exiting the collimating lens depends on the distance of the bundle distal end to the collimating lens, as well as the diameter of the bundle and collimating lens, the f-number of the lens and the numerical aperture of the bundle. By way of example, when the diameter of the bundle <b>62</b> is in the range of about 6.3 mm to 6.4 mm, the diameter of the collimating lens <b>67</b> is in the range of about 16.5 mm to about 17 mm, the f-number is one and the distance between the optical output end <b>62</b> and the collimating lens <b>67</b> is in the range of about 19 mm to about 20 mm, a substantially skewed light ray exiting the collimating lens would be one in the range of greater than about 14 degrees from the direction substantially parallel to the longitudinal axis of the perpendicular to the optical output end <b>62</b>.

0066In accordance with another aspect of the present invention as is best illustrated in <figref idref="DRAWINGS">FIG. 6</figref>, a light baffle device, generally designated <b>70</b>, is deployed between the optical output end <b>62</b> and the collimating lens <b>67</b> to intercept light these skewed light rays. Accordingly, the baffle device <b>70</b> will substantially prevent skewed rays from reflecting off of interior walls and entering the collimating lens <b>67</b> and thus leak around the filter <b>63</b>.

0067The light baffle device <b>70</b>, in one embodiment, includes an opaque plate member <b>75</b> disposed substantially adjacent an upstream abutting surface <b>71</b> of the collimating lens. Centrally disposed in the plate member is an aperture <b>72</b> extending there through, and having a transverse cross-sectional area smaller than that of the collimating lens abutting surface <b>71</b>. Preferably, the ratio of the transverse cross-sectional area of the aperture <b>72</b> to that of the abutting surface <b>71</b> of the collimating lens <b>67</b> is in the range of about 0.64:1 to about 0.8:1.

0068Thus, the skewed light rays impinging upon the upstream surface <b>73</b> of the plate member <b>75</b> are intercepted, while the remaining portion of the light transmitted from the first bundle portion <b>50</b> pass through aperture <b>72</b>. Further, to reduce reflection of these impinging light rays, the plate member <b>75</b> is either coated with a material which absorbs light, such as black anodize, or is composed of opaque materials having absorption properties, such as black Delrin.

0069In the preferred form, the aperture <b>72</b> of the baffle device <b>70</b> is substantially central to the abutting surface <b>71</b> of the collimating lens <b>67</b>. Thus, a longitudinal axis of the aperture <b>72</b> is substantially co-axially aligned with a longitudinal axis of the collimating lens <b>67</b>.

0070In still another configuration, a plurality of plate members <b>75</b>_A-<b>75</b>_Dare nested together in a side-by-side manner in abutment with the collimating lens abutting surface. Briefly, while four plate members <b>75</b>_A-<b>75</b>_Dare shown and described, it will be appreciated that more or less plate members may be applied.

0071<figref idref="DRAWINGS">FIGS. 6 and 7</figref> further illustrates that each plate member <b>75</b>_A-<b>75</b>_Ddefines a respective central aperture <b>72</b>_A-<b>72</b>_Dwhich is co-axially aligned with the longitudinal axis of the abutting surface <b>71</b> of the collimating lens. A threaded ring <b>76</b> or the like is deployed in the input port <b>58</b> and matably engaged with the first connector <b>61</b> to affix the plate member <b>75</b>_A-<b>75</b>_Dagainst the abutting surface <b>71</b> of the collimating lens <b>67</b>. Further, each aperture <b>72</b>_A-<b>72</b>_Dhas a respective transverse cross-sectional area smaller than that of the collimating lens abutting surface <b>71</b>. However, each adjacent downstream plate member <b>75</b>_B-<b>75</b>_Ddefines a respective aperture <b>72</b>_B-<b>72</b>_Dhaving a diameter incrementally larger than its adjacent upstream plate member <b>75</b>_A-<b>75</b>_C. Preferably, the area of each successive downstream aperture <b>72</b>_B-<b>72</b>_Dis about 10% to about 25% larger.

0072Each aperture <b>72</b>_A-<b>72</b>_Dfurther tapers outwardly in a direction toward the collimating lens <b>67</b> such that a respective entrance diameter (D_en) of the respective aperture <b>72</b>_A-<b>72</b>_Dis smaller than the corresponding exit diameter (D_ex) thereof. Preferably, the outward taper of each aperture is in the range of about 30° to about 60° from the longitudinal axis, and the thickness of each plate member is in the range of about 0.5 mm to about 2.0 mm.

0073However, in accordance with the present invention, while the overall transverse cross-sectional dimension of each successive downstream aperture <b>72</b>_B-<b>72</b>_Dis progressively larger, the entrance Diameter (D_en) of the corresponding downstream aperture <b>72</b>_B-<b>72</b>_Dof the plate member <b>75</b>_B-<b>75</b>_Dis smaller than the exit Diameter (D_ex) of the adjacent aperture <b>72</b>_A-<b>75</b>_Cof the adjacent upstream plate member <b>75</b>_A-<b>75</b>_C. Accordingly, if this nested configuration of the plate members were provided as an integral single unit, a plurality of annular ribs and adjacent troughs would be defined with each annular rib being successively larger in diameter as the plate member is positioned closer to the collimating lens <b>67</b>.

0074This is beneficial in that should a sufficiently skewed light ray pass through the upstream aperture <b>72</b>, it may be intercepted in the annular trough between two adjacent ridges.

0075In another aspect of the present invention, a light-tight fiber optic connection assembly, generally designated <b>52</b>, is included for optically connecting the distal transmission end <b>77</b> of second bundle portion <b>51</b> to the proximal receiving end <b>78</b> of third bundle portion <b>53</b>. This optical connector assembly <b>52</b>, as best viewed in <figref idref="DRAWINGS">FIGS. 2</figref>, <b>4</b>, <b>8</b> and <b>9</b>, includes a connector body <b>80</b> mounted to a side wall <b>85</b> of the imaging apparatus <b>21</b> for optical communication into the light-tight imaging compartment <b>25</b>.

0076The connector body <b>80</b> includes a proximal portion <b>81</b> and an opposite distal portion <b>82</b>, and defines an elongated passage <b>83</b> extending from the proximal portion <b>81</b> to the distal portion <b>82</b>. The proximal portion <b>81</b> of the connector body is adapted to removably couple to a distal optical connector <b>86</b> mounted to the second bundle portion <b>51</b>, while the distal portion <b>82</b> of the connector body is adapted to removably couple to a proximal optical connector <b>87</b> mounted to the third bundle portion <b>53</b>. Each of the distal optical connector <b>86</b> and the proximal optical connector are conventional female optical connectors which are threaded to the corresponding threaded male end of the connector body.

0077When the optical connectors <b>86</b>, <b>87</b> are properly mounted to the connector body <b>80</b>, the transmission ends <b>77</b> of the fiber optic strands of the second bundle portion <b>51</b> terminate in the passage <b>83</b> in opposed relationship to the receiving ends <b>78</b> of the fiber optic strands of the third bundle portion <b>53</b>. A diffuser device <b>88</b> is further disposed in the passage <b>83</b> in the gap region <b>90</b> between the second bundle transmission end <b>77</b> and the third bundle receiving end <b>78</b>. This diffuser device <b>88</b> is adapted to diffuse the light transmitted from the transmission end of the fiber optic strands to facilitate receipt in the receiving end.

0078As the collective light is diffused while passing through diffuser device <b>88</b>, it is substantially uniformly distributed about the proximal receiving ends <b>78</b> of the third bundle portion <b>53</b>. Consequently, the light ring mounted peripherally about the view port <b>23</b> uniformly illuminates the specimen platform <b>26</b>. Without this diffuser, the collimated and filtered light transmitted across the gap may develop “hot-spots” on the proximal receiving end <b>78</b> of the third bundle portion <b>53</b> which causes distribution non-uniformity from the light ring.

0079The diffuser may be any substantially transparent device capable of uniform diffusion of the light passing there through. Preferably, the diffuser device <b>88</b> is composed of a rigid material, such as plastic or glass which is seated across the transverse cross-sectional dimension of the connector body passage <b>83</b>. To provide uniform light diffusion, at least one surface of the diffuser device <b>88</b> upon which the light must pass through is frosted. Typical of these diffusers, by way of example, is model L45-652, by Edmund Scientific of Barrington, N.J.

0080As illustrated in <figref idref="DRAWINGS">FIG. 9</figref>, the diffuser device <b>88</b> is preferably plate-like having substantially flat, opposed surfaces, of which at least one is frosted. Preferably, an interior wall <b>91</b> of the connector body, defining the passage <b>83</b>, is substantially cylindrical-shaped. An insert sleeve <b>89</b> having an outer diameter substantially similar to but smaller than that of the passage <b>83</b> is slideably inserted between the transmission ends <b>77</b> and the receiving ends <b>78</b> of the strands in the gap region <b>90</b>. The insert sleeve <b>89</b> includes an annular shoulder portion <b>92</b> upon which the outer circumferential edge of diffuser device <b>88</b> seats. When the insert sleeve <b>89</b> is slideably disposed in the gap region <b>90</b> of the passage <b>83</b>, the opposed surfaces of the diffuser device <b>88</b> extend across the entire transverse cross-sectional dimension of the passage <b>83</b> to assure the light transmitted from the distal end of the second bundle portion <b>51</b> of the fiber optic strand bundle passes through the diffuser device.

0081By way of example, for a fiber optic bundle with a diameter of about 12.6 mm to about 12.7 mm, the connector body passage <b>83</b> may have a diameter in the range of about 12.7 mm to about 12.8 mm. Upon mating of the optical connectors to the connector body, the gap between the opposed ends of the second bundle portion <b>51</b> and the third bundle portion <b>53</b> may be in the range of about 8.0 mm to about 15.0 mm. The diffuser device <b>88</b> accordingly, may have a thickness in the range of about 1.0 mm to about 2.2 mm.

0082Referring now to <figref idref="DRAWINGS">FIGS. 10 and 11</figref>, an emission filter wheel <b>96</b> is housed in the imaging chamber <b>25</b> to filter out the excitation light rays, leaving substantially only the fluorescent rays emanating from the sample for capture by the CCD camera <b>31</b>. Depending upon which excitation filter is necessary in the excitation filter wheel <b>57</b> to excite the desired fluorescent spectra in the imaging compartment <b>25</b>, a corresponding emission filter <b>95</b> is selected to absorb the excitation rays and allow passage of the fluorescent emission into the lens <b>97</b> of the camera <b>31</b>.

0083In one specific embodiment, emission filter wheel <b>96</b> is rotatably mounted to the upper housing <b>22</b> of the imaging apparatus <b>21</b>, between the frame <b>32</b> of the light ring <b>30</b> and lens <b>97</b> of the camera <b>31</b> (FIG. <b>11</b>). As best viewed in <figref idref="DRAWINGS">FIG. 10</figref>, the filter wheel includes a plurality of filters each of which can be selectively rotated about optical filter wheel axis <b>98</b>, and into optical alignment with the aperture of the light ring <b>30</b>.

0084Due to size constraints and limitations, a crescent-shaped gap <b>100</b> may be formed between the outer peripheral edge <b>101</b> of the frame <b>99</b> of the filter wheel <b>96</b> and the view port <b>23</b> of the upper housing <b>22</b> (FIG. <b>10</b>). While this gap <b>100</b> is not large, it is sufficient to permit the passage of some unfiltered light rays along the path of arrow <b>102</b> in FIG. <b>11</b>. Accordingly, to prevent this light leakage, a light-tight seal device <b>105</b> is positioned between the filter wheel frame <b>99</b> and the upper wall <b>103</b> of the upper housing.

0085This seal device <b>105</b> is sufficiently dense and/or opaque to prevent the passage of light there through yet sufficiently flexible to allow relative rotation of the second filter wheel without exerting undue stress thereon. Preferably, the seal device <b>105</b> is provided by a pair of brush devices positioned in the gap. One such brush material is a plastic brush, by Amesbury Group, Inc. of Statesville, N.C.

0086Referring now to <figref idref="DRAWINGS">FIGS. 12-14</figref>, <b>18</b> and <b>19</b>, another specific embodiment of the macroscopic fluorescence illumination assembly <b>28</b> is illustrated. In this configuration, the illumination assembly <b>28</b> includes a fluorescent light source <b>37</b>, and a light dispersion assembly <b>110</b> positioned proximate the view port <b>23</b> of the interior wall <b>103</b>. The illumination assembly <b>28</b> further includes a bundle <b>111</b> of fiber optic strands composed of substantially pure fused silica. The proximal ends <b>112</b> thereof in optical communication with the light source <b>37</b> and distal ends <b>113</b> thereof terminate proximate the view port <b>23</b>. The distal ends <b>113</b> each emit a conical directional beam of light originating from the light source <b>37</b> and cooperating with the light dispersion assembly <b>110</b> such that the plurality of directional beams <b>115</b> (shown in phantom lines) collectively illuminate the specimen platform <b>26</b> in a substantially uniform manner.

0087As mentioned above, all materials (glasses, etc.) fluoresce at some level. Ordinary optical glass materials applied for fiber optic strands contain impurities that can fluoresce. It has been observed that high purity or substantially pure fused silica exhibits very low autofluorescence. This is of course beneficial to reduce undesirable autofluorescence of the fiber optic strand material which passes through the emission filter and may be mistaken for “sample” fluorescence. In particular, the fiber optic strands and filters are composed of as plastic clad fused silica or silica clad fused silica.

0088High purity or substantially pure fused silica is less flexible than glass or other conventional materials applied for fiber optic strands. Thus, the permissible bending radius or radius of curvature of a bundle of fiber optic strands (i.e., the minimum suggested usable bending radius of the fiber optic bundle without fracture of the strands) composed of such fused silica is substantially greater than that for conventional fiber optic strand materials. A fused silica core/clad diameter must be significantly reduced to obtain the same radius of curvature. Thus, about twice as many fused silica fibers must be used to transmit the same amount of light, and the cost becomes a significant factor.

0089In this specific embodiment, since the bundle of fiber optic strands originates from an interior side wall <b>85</b> of the imaging apparatus <b>21</b>, as shown in <figref idref="DRAWINGS">FIG. 12</figref>, similar to the light-ring embodiment above, the optical axes of the bundle are contained in a generally horizontal plane which is generally parallel with that of the specimen platform <b>26</b>. Thus, the optical axis of the distal ends of the strands needs to be redirected and repositioned in a direction toward the specimen platform of illumination thereof. However, due to the relatively large radius of curvature of the substantially pure fused silica strands, the overall vertical footprint of the imaging apparatus and costs are significantly increased.

0090To address this problem, the dispersion assembly <b>110</b> is configured to cooperate with the distal ends <b>113</b> of the fiber optic strands to redirect the directional beams <b>115</b> (shown in phantom lines) collectively toward the specimen platform <b>26</b> for illumination thereof in a substantially uniform manner. Accordingly, the optical axes of the distal ends <b>113</b> of the fiber optic strands may be retained generally parallel to the specimen platform <b>26</b>, while the directional beams are directed (E.g., through reflective surfaces <b>116</b>) downwardly toward the specimen platform <b>26</b>. The overall height of the imaging apparatus <b>21</b>, thus, is significantly reduced since the distal ends of the substantially pure fused silica fibers themselves need not be curved toward the platform <b>26</b>, and the overall cost is significantly reduced.

0091Referring now to <figref idref="DRAWINGS">FIGS. 15 and 16</figref>, the light dispersion assembly <b>110</b> includes a bracket device <b>117</b> adapted to mount and secure the distal ends <b>113</b> of the fiber optic strands to the upper interior wall <b>103</b> of the imaging apparatus <b>21</b>. These bracket devices <b>117</b> are preferably substantially rigid, and are composed of black anodized aluminum to reduce auto fluorescence.

0092In one specific embodiment, to redirect the directional beams emitted from each distal end <b>113</b> of the strands, the dispersion assembly <b>110</b> includes a reflective surface <b>116</b> angled to reflect the directional beams toward the specimen platform <b>26</b>. This permits the entire fiber optic bundle <b>111</b> to be maintained in generally the same plane which is essentially parallel to the specimen platform <b>26</b>.

0093To reflect the directional beams about 90° from the optical axis of the distal ends of the strands and toward the specimen platform, the relatively planar reflective surface <b>116</b> should be oriented about 45° relative the direction of the optical axis. It will be appreciated that depending upon the particular position of the bracket device <b>117</b> and the exact orientation of the optical axis from the relative the desired position along the specimen platform to be illuminated, the angle of the reflective surface can be altered accordingly.

0094In one application, illumination “hot spots” can be reduced by diffusing the directional beams as they reflect off of the reflective surface <b>116</b>. This improves the light distribution across the specimen platform so that the illumination is substantially uniform. One diffuser technique is to provide a diffusing surface <b>114</b> which cooperates with the reflective surface <b>116</b> to uniformly diffuse the directional beams emitted from the strand distal ends <b>113</b>. For example, the reflective surface <b>116</b> may be provided by an aluminum plate with a roughened surface or by SPECRALON®, which diffuses the reflected light as it impinges the surface thereof.

0095Another diffuser technique is to provide slightly convex reflective surface configured to provide substantially uniform illumination across the specimen platform <b>26</b>. Other conventional diffuser techniques or a combination thereof may be employed without departing from the true nature and scope of the present invention. Moreover, the desired diffusion pattern after reflection off of the reflective surface is dependent in-part on the particular shape and radius of the convex reflective surface, the distance of the surface from the reflective surface, and the distance from the reflective surface to the specimen platform.

0096Each bracket device <b>117</b> includes a mounting section <b>118</b> adapted to removably secure the distal ends <b>113</b> of the fiber optic bundle in a manner directing their emitted directional beams of light against the reflective surface <b>116</b>. As best viewed in <figref idref="DRAWINGS">FIGS. 15 and 16</figref>, the mounting section defines a passage <b>120</b> which is formed and dimensioned for sliding receipt of the fiber optic bundle <b>111</b> there through. An adjustable fastener <b>121</b> affixes the bundle to the bracket device to enable the distal ends <b>113</b> of the fiber optic strands to be displaced closer to or further away from the reflective surface <b>116</b> during calibration. In one example, the fastener may be simply be a set screw or the like.

0097<figref idref="DRAWINGS">FIG. 16</figref> best illustrates that the distal ends <b>113</b> of the strands are positioned relatively close to the reflective surface <b>116</b> of the mirror, without actual contact therewith. By positioning the distal ends relatively close to the reflective surface, the diffusion of the reflected directional beams are better controlled to be substantially within the specimen platform <b>26</b> boundaries. However, it should be appreciated that the distal ends should not be too close or in contact with the reflective surface so as to minimize reflection back through the strand distal ends <b>113</b>.

0098For example, the distal ends of the fiber optic strands, in one specific configuration, are positioned in the range of about 6 mm to about 11 mm from the reflective surface, and more preferably about 8 mm. In this example, the reflective surface <b>116</b> of the reflector <b>122</b> is positioned in the range of about 120 mm to about 460 mm from the specimen platform. Thus, the collective vertical footprint of this configuration is significantly reduced by applying this dispersion assembly.

0099Preferably, the dispersion assembly includes a plurality of bracket devices <b>117</b> peripherally spaced about the view port <b>23</b> of the interior wall <b>103</b>. Such peripheral spacing even more uniformly distributes the reflected directional beams about the view port <b>23</b>, and further reduces shadowing. Four (4) to eight (8) bracket devices <b>117</b> have been found sufficient to assure illumination uniformity, but more or less may be applied as well.

0100Referring now to <figref idref="DRAWINGS">FIGS. 12 and 17</figref>, the illumination assembly <b>28</b> may include a protective cover device <b>123</b> (shown in phantom lines in FIG. <b>17</b>). This cover provides protection for the lighting components as well as reduce the residual auto fluorescence of the lighting components. The protective cover device <b>123</b> includes corresponding apertures <b>124</b> (<figref idref="DRAWINGS">FIG. 12</figref>) each aligned with the respective reflective surface <b>116</b>, and extending through the bottom wall of the cover device to permit passage of the reflected directional beams. Further, a larger aperture <b>125</b> corresponding to and aligned with the view port <b>23</b> is provided to enable passage of the light to the camera.

0101As shown in <figref idref="DRAWINGS">FIGS. 18 and 19</figref>, the filter assembly <b>47</b> may include a mounting bracket (not shown) which affixes the housing <b>57</b> to one wall of the imaging apparatus. The side wall <b>85</b> of the housing <b>57</b> includes a passage <b>126</b> sized for sliding receipt of the downstream connector portion <b>127</b> of the filter housing <b>57</b> there through. A threaded nut <b>128</b> may also be provided which is fastened from inside the imaging chamber <b>25</b> to affix the filter assembly <b>47</b> to the imaging apparatus. The proximal connector end <b>130</b> of the fiber optic bundle <b>111</b> can then be removably attached to the downstream connector portion.

0102This arrangement is particularly suitable for this application since the length of the fused silica clad silica fiber optic bundle can be minimized. In effect, the path length of the filtered light passing through the fused clad silica fiber optic bundle is reduced significantly to minimize any auto fluorescence thereof, even though such material already has low auto fluorescence properties. Moreover, high purity or substantially pure fused clad silica is very expensive, and minimizing the length of the bundle ultimately reduces costs.

0103Referring now to <figref idref="DRAWINGS">FIGS. 20-24</figref>, in still another aspect of the present invention, an alternative embodiment macroscopic fluorescence illumination assembly, generally designated <b>140</b>, is provided for use with the imaging apparatus <b>21</b>. In this configuration, a bottom illumination configuration is provided that significantly reduces background fluorescent or autofluorescent signals emitted from the endogenous animal tissue itself. Briefly, this is one of the most significant limitations to fluorescent imaging contrast. Biological tissues have an inherent fluorescent signal that is strongly excited by excitation light that is in the blue/green region of the spectrum. These autofluorescent signals limit the sensitivity of detection by reducing the signal to background of the desired fluorescent labeling reagent.

0104There are several techniques that can reduce this background fluorescent signal, one of which includes moving the excitation light source wavelength towards the infrared region of the spectrum. In this region, the tissue autofluorescence is reduced, but not totally eliminated. The drawback to this technique is that it is not always feasible to use an infrared dye which would be necessary. Another approach to minimize the tissue autofluorescence signal from the sample is to alter the illumination configuration. The illumination design in the above-mentioned configuration is that of a front illumination profile, sometimes referred to as top illumination, where the excitation light source in the fluorescent kit and the detection equipment (i.e., camera <b>31</b>) are on the same side of the specimen. Since the excitation light intensity is highest on the side of the specimen facing the camera <b>31</b>, the tissue autofluorescent signal emitted is also highest on this side. Thus, the ratio of the signal to background noise of the desired fluorescent labeling reagent is significantly reduced, limiting the sensitivity of detection by the camera.

0105In accordance with the bottom illumination assembly design of the present invention, the excitation light source is located on the opposite side of the specimen as the detection system. Generally referred to as transillumination or bottom illumination, this illumination configuration reduces the autofluorescent signal from the sample by trapping the autofluorescent light on the opposite or bottom side of the animal relative to the camera.

0106Referring to <figref idref="DRAWINGS">FIGS. 20</figref>, <b>21</b> and <b>23</b>, the bottom side illumination assembly <b>140</b> is shown including a specimen support surface <b>141</b> sized and dimensioned for receipt in the imaging compartment <b>25</b> atop the specimen platform <b>26</b> of the imaging apparatus <b>21</b> (e.g., as shown in FIG. <b>1</b>). The support surface <b>141</b> is substantially opaque and defines a window portion <b>142</b> that enables the passage of light there through which is oriented to face toward the view port <b>23</b> thereof. The window portion is selectively sized and dimensioned such that when the specimen is supported atop the support surface <b>141</b>, it can be positioned and seated fully over the window portion in a manner forming a light-tight seal substantially there between. The illumination assembly <b>140</b> further includes an excitation light source <b>37</b>, and a bundle of fiber optic strands <b>143</b> having proximal ends thereof in optical communication with the light source <b>37</b>. The distal ends of the strands terminate proximate the window portion of the support surface. The distal ends each emit a respective beam of light originating from the light source <b>37</b> which are then collectively directed toward the window portion <b>142</b> and into a bottom side of the specimen <b>145</b>.

0107Accordingly, since biological tissue is a turbid medium, the excitation light entering the specimen becomes multiply scattered and diffused where it propagates throughout the entire specimen, thereby exciting the fluorophore. The fluorophore emission is also scattering by tissue, and eventually, some of the scattered excitation light exits the topsides of the specimen <b>145</b>, and is captured through the view port <b>23</b> and into the camera <b>31</b>.

0108In one specific configuration, the bottom illumination assembly <b>140</b> includes a specimen illumination platform, generally designated <b>146</b>, having a support structure <b>147</b> and a cover plate <b>150</b> removably mounted atop the support structure. The support structure <b>147</b> is preferably rectangular shaped having four upstanding side walls <b>151</b> surrounding an interior cavity <b>152</b> thereof. The support structure <b>147</b> is preferably substantially rigid, and may be fabricated from one or more pieces of black anodized aluminum, however, to reduce auto fluorescence. Other rigid materials may be applied, as noted above. The interior surface of the upstanding walls <b>151</b> are preferably coated with a material which absorbs light, such as black anodize, or is composed of opaque materials having absorption properties, such as black Delrin. It will further be appreciated, of course, that the support structure can be other shapes as well.

0109Mounted atop the upper edges of the upstanding walls <b>151</b> is the cover plate <b>150</b> that incorporates the support surface <b>141</b> to support the specimen <b>145</b>. The cover plate <b>150</b> is also preferably composed of a rigid material such as black anodized aluminum to reduce autofluorescence. The peripheral footprint of the cover plate is rectangular, and is sized to match that of the support structure <b>147</b> when mounted thereto. A plurality of set screws <b>156</b> (eight as shown) cooperate with aligned screw holes <b>157</b>′, <b>157</b> (<figref idref="DRAWINGS">FIG. 24</figref>) in the respective cover plate <b>150</b> and support structure <b>147</b> to removably mount the two together. A gasket or the like may be provided between the interface to assure a light-tight seal there between.

0110Extending through the cover plate from the support surface <b>141</b> to a bottom side is an aperture <b>153</b> that enables the excitation light to pass from the interior cavity <b>152</b>, and into the specimen. Thus, in some configurations, the aperture <b>153</b> functions as the window portion <b>142</b> of the support surface <b>141</b>. This aperture is preferably rectangular shaped, but can be any size and/or shape to better coordinate with the shape of the specimen supported over the aperture. When the aperture <b>153</b> functions as the window portion, the specimen must be large enough to form a light-tight seal all around the edge of the aperture <b>153</b> when it is properly seated atop the support surface <b>141</b>. Thus, essentially, the peripheral footprint of the aperture <b>153</b> must be sufficiently smaller than that of the properly oriented specimen <b>145</b> to form such a seal. It will be understood that without the formation of this light-tight seal between of the specimen with the edge defining the aperture, unscattered excitation light would leak into the imaging compartment <b>25</b> of the imaging apparatus <b>21</b> and be detected by the sensitive camera <b>31</b>.

0111In one specific example, the opening (i.e., window portion <b>142</b>) upon which light may pass through from the interior cavity <b>152</b> of the specimen illumination platform <b>146</b>, and into the specimen can be sized and dimensioned to assure the formation of such a light-tight seal. This may be performed by providing a removable slide device <b>155</b> (<figref idref="DRAWINGS">FIGS. 23 and 24</figref>) or the like that is sized for aligned positioning into a receiving slot <b>158</b> formed in the support surface <b>141</b> of the cover plate <b>150</b>. This receiving slot <b>158</b> is generally rectangular shaped, and is of a height profile such that when the slide device <b>155</b> is received in the receiving slot <b>158</b>, the top surface of the slide device <b>155</b> is substantially flush with the top surface of the cover plate <b>150</b>, thus becoming the support surface <b>141</b>.

0112The slide device <b>155</b> is preferably formed of a transparent material, such as a glass slide, sized and dimensioned for receipt in the receiving slot <b>158</b> (FIGS. <b>21</b> and <b>24</b>). By painting the top or bottom side of the glass with an opaque paint material, such as Krylon black paint, the transparent window portion <b>142</b> that permits the passage of the excitation light into the specimen can be formed from a void in the painted surface. Using a plurality or set of slide devices <b>155</b>, a variety of different size and shape window portions <b>142</b> can be provided. Accordingly, the size of the window portion <b>142</b> can be altered by merely changing the slide device <b>155</b>. As mentioned, should the specimen have a footprint large enough, the slide device <b>155</b> can be removed altogether and the specimen be placed directly atop and over the aperture <b>153</b>.

0113To assure cleanliness of the slide device <b>155</b>, a disposable lining (not shown) may be included which is positioned atop the support surface <b>141</b> of the slide device. This disposable lining, which may be composed of paper, is preferably dimensioned to seat into and along the peripheral lining edge <b>160</b> surrounding the receiving slot <b>158</b> of the cover plate <b>150</b>. This lining edge performs the function of aligning and seating the disposable lining, similar to that between the receiving slot <b>158</b> and the slide device <b>155</b>. The lining includes an opening larger than the window portion <b>142</b> of the slide device <b>155</b> so as not to obstruct the passage of excitation light into the specimen.

0114Similar to the top illumination configuration above-mentioned, the bottom illumination assembly <b>140</b> includes a bundle <b>143</b> of fiber optic strands composed of materials selected to have low autofluorescence properties. Again, one material particularly suitable for the fiber optic strands and filters is high purity fused silica, such as plastic clad fused silica or silica clad fused silica, which has very low autofluorescence. Further, as indicated above, all exterior fiber optic bundle portions of the fiber optic bundle <b>35</b> include a segmented, flexible metal jacket (not shown) placed around these portions together with an exterior flexible PVC sleeve <b>42</b> to reduce the introduction of exterior light or noise into the fiber optic strands. This sleeve is opaque (black), and blocks all external ambient light that might enter the fiber bundle.

0115The fiber optic bundle portions internal to the imaging box are surrounded by a non-phosphorescent material to substantially eliminate the possibility of spurious light sources from the fiber optic bundle within the imaging box. One particular flexible material which has low phosphorescence is a polyolefin heat shrink tubing material. Again, all optical filters (e.g., interference-type filters including bandpass filters, longpass filters and shortpass filters), filter wheel assemblies, illumination sources, fiber optic connections external to and into the imaging apparatus <b>21</b> are similar to that discussed above. Further, while the composition of the fiber optic strands for the internal fiber optic bundle may be composed of fused silica, as mentioned above, they are preferably composed of conventional fiber optic glass. Relatively costly fused silica has lower autofluorescence properties, but this is not as critical for bottom illumination where the autofluorescence is trapped under the specimen where it is not detected or seen by the camera.

0116In this specific embodiment, the bundle of fiber optic strands for the bottom illumination assembly <b>140</b> originates from an interior side wall <b>85</b> of the imaging apparatus <b>21</b> similar to the top illumination assemblies such as the light-ring embodiment above. In the bottom illumination assembly <b>140</b>, however, the optical connector into the imaging apparatus <b>21</b> may be positioned lower to the bottom specimen platform <b>26</b> so that the bundle of fiber optic strands can extend into the interior cavity <b>152</b> of the support structure <b>147</b> without being significantly bent.

0117<figref idref="DRAWINGS">FIGS. 22 and 24</figref> best illustrates that the distal ends <b>161</b> of the fiber optic bundle <b>143</b> terminate in the interior cavity <b>152</b> of the specimen illumination platform <b>146</b>. A bundle slot <b>162</b> is provided in one of the upstanding walls <b>151</b> of the support structure <b>147</b> for receipt of the fiber optic bundle portion <b>143</b> there through. In one configuration, the distal ends <b>161</b> of the fiber strands of the fiber optic bundle <b>143</b> are oriented to direct the conical beams of light emitted there from directly through the window portion <b>142</b> and into the specimen seated thereatop.

0118Similar to the dispersion assembly <b>110</b> of the top illumination assembly above, a reflector device <b>163</b> is included in the interior cavity <b>152</b> of the support structure <b>147</b> that is configured to cooperate with the distal ends <b>161</b> of the fiber optic strands to redirect the directional beams collectively through the window portion <b>142</b> of the slide device <b>155</b>. Accordingly, the optical axes of the distal ends <b>161</b> of the fiber optic strands may be retained generally parallel to the horizontal plane of the fiber optic bundle portion extending through the bundle slot <b>162</b> and into the interior cavity <b>152</b> of the support structure <b>147</b>, while the directional beams emitted from the strand distal ends are reflected (E.g., through reflector device <b>163</b>) upwardly through the window portion <b>142</b> and into the specimen <b>145</b>. The overall height of the bottom illumination assembly <b>140</b> can, thus, be significantly reduced since the distal ends of the fibers themselves need not be curved upward toward the window portion.

0119The reflector device <b>163</b> includes a reflective surface <b>165</b> oriented at an angle about 45° relative the direction substantially parallel to the optical axes of the distal ends of the fiber optic strands to reflect the directional beams of light about 90° from that emitted from the distal ends. To orient the reflector device <b>163</b> properly relative the distal ends <b>161</b> of the fiber optic bundle, mounting supports <b>166</b> are provided in the interior cavity <b>152</b> of the support structure upon which a backside of the reflector device seats. The reflector device <b>163</b> is then either permanently seated against the mounting supports through an adhesive, or removably mounted to permit interchangeability of the reflector device.

0120The reflective surface <b>165</b> is preferably mirror-like for non-diffuse reflection of the excitation light fully into the bottom side of the specimen. In this configuration, the turbid medium of the biological tissue is relied upon to multiply scatter and uniformly propagate the excitation light though the entire specimen. In other configurations, the reflective surface <b>165</b> may include a diffuse material that diffuses the light reflected through the window portion <b>142</b>. As mentioned above, these diffuser materials may include a roughed aluminum surface or SPECRALON®.

0121The reflective surface <b>165</b> is preferably substantially planar. It is conceivable, however, that the reflective surface could be partially concave to reflectively focus the reflected excitation light toward a smaller area or through discretely shaped window portions. This would be particularly valuable should the size and shape of the window portion be smaller than the size and shape of the layout distribution of the distal ends of the fiber optic bundle. In the configuration shown in <figref idref="DRAWINGS">FIGS. 22 and 24</figref>, however, the reflective surface <b>165</b> is preferably rectangular-shaped. Both the reflective surface <b>165</b> and the layout distribution of the distal ends of the fiber optic bundle, as will be described, are sized and dimensioned to match one another. Accordingly, both the reflective surface and the layout distribution of the distal ends of the fiber optic bundle <b>143</b> are elongated and rectangular-shaped (i.e., conforming to the rectangular-shaped aperture <b>153</b> and window portion <b>142</b>).

0122As shown, the distal ends of said fiber optic bundle are thus aligned in a linear array extending substantially along the elongated reflective surface <b>165</b>. Preferably, the distal ends <b>161</b> in the fiber optic cable are rearranged from a round bundle (about 0.7 cm in diameter) to a line or an elongated rectangle array of fibers that is approximately 6 cm long and 0.2 cm wide. One example of a round to linear fiber cable is the distribution unit <b>167</b> that commercially available from Dolan Jenner Industries of Lawrence, Mass., model no. BL405. In this example, the interior cavity <b>152</b> of the support structure <b>147</b> is sized to slideably receive the rectangular housing of the distribution unit <b>167</b> in a manner automatically aligning and spacing the distal ends <b>161</b> along and relative to the reflector device <b>163</b>.

0123Although only a few embodiments of the present inventions have been described in detail, it should be understood that the present inventions may be embodied in many other specific forms without departing from the spirit or scope of the inventions.